Among helminths, digenetic trematodes comprise more than 100 families. Most trematodes are relatively little aggressive parasites living in intestines and other organs of vertebrates; thus, they have been receiving little attention from parasitologists that make use of applied parasitology. Trematodes known by causing serious diseases to humans, i.e. blood stream trematodes, Schistosoma, as well as liver and lung trematodes are very important animal-infecting parasites.
Schistosomiasis is a disease caused by blood trematodes belonging to family Schistosornatideae, class Trematoda, subclass Diginea, and gender Schistosoma. Humans are mainly parasitized by three parasite species of the gender Schistosoma: S. mansoni (found in Africa and South America), S. haematobium (Africa and Middle East), and S. japonicum (Asia). Adult worms of S. mansoni and S. japonicum are located in mesenteric veins of intestines, while S. haematobium occurs in veins surrounding the bladder.
Fasciola, the most important liver trematode, is the predominant parasite in domestic ruminants and it is responsible for serious economical losses over the world, reaching the bovine, caprine, and ovine cattle.
The main disease characteristic, responsible for the pathology, morbidity and mortality of the cited animals, is based on the host's hepatic tissue destruction, as a result of the damages caused to bile ducts, where the adult specimen Fasciola lives. Young animals, particularly infected by Fasciola hepatica, have higher morbidity and consequently they die. Fasciola, some times, can also parasitize humans, when an opportunity of it enters in contact with the habitat of the animal disease occurs. This fact is more frequent in Cuba and some countries of Latin America. However, the real human's liver trematode is another parasite called Clonorchis sinensis, which is pervasive in China, Japan, Korea, Vietnam, and India.
Basically, the pathology is caused by the thickening of the bile duct walls and, in more severe cases, may cause liver cirrhosis and death.
Both Fasciola and Clonorchis enter passively into the host, under a larval form called metacercaria that is ingested with food (pasture and raw fish for Fasciola and Clonorchis, respectively); however, their pathway of migration into vertebrate host organism occurs through bile ducts and they differ amongst themselves. While Clonorchis reaches the bile tree through intestines, and ampulla of Vater; Fasciola migrates through abdominal cavity, penetrating actively into the liver wall, by its capsule, reaching the parenchyma and the bile system; thus, causing serious damages to host's tissues.
Regarding the fascioliasis in pets, there are conflicting results and little evidence suggesting that sheep or goats acquire immunity against Fasciola hepatica (Sinclair, 1967) after immunization with raw extracts.
There is evidence showing that the infection may persist for at least 11 years in sheep experimentally infected (Durbin, 1952). It was also reported very little or no reaction of the host to parasite; thus, the infected sheep survival will depend entirely on the number of metacercaria ingested (Boray, 1969). The bovine cattle are known to be more resistant against F. hepatica. Fasciola in these cases usually lives in host for an average of 9-12 months; however, young animals are the most clinically affected by fascioliasis.
In order to identify the antigens that can be used for immunoprophylaxis and that could serve as a basis for developing vaccines effective against fascioliasis, several attempts have been made. Several scientists have been following basically two independent experimental strategies based on: 1) immunity induced by irradiated living cercaria, the basis of the considered living vaccine and, 2) immunity induced by the called non-living vaccines.
However, few works have been published in the context of obtaining acquired resistance to Fasciola hepatica in calves by using somatic raw extracts from the adult parasite. Ross, 1967, Hall and Lang, 1978, Hillyer, 1979 have reported conflicting data in this context.
The immunity induced by vaccines obtained from irradiation of metacercaria, i.e. irradiated or attenuated living or non-living vaccines, also has lead to frustrating results in experiments performed with mice, rabbits and sheep (Campbell et al, 1978, Hughes, 1963), since no evidence of the development of important immunity has occurred after the irradiated metacercaria administration in these animals.
Additionally, experiments with distinct extracts from excretion and secretion products of trematodes, in their adult form, obtained directly from bile ducts, have been shown to be non-immunogenic since the animals vaccinated with materials originating from these parasitic forms, similarly to controls of the experiments, presented very low or no protection and lesions, from the pathological point of view, in the liver parenchyma.
It is expected, on the state-of-the-art basis, that the bovine cattle can respond better to vaccination with non-living vaccines. For the caprine cattle, there are no experimental indications suggesting that similar situation could be anticipated based on merely mediocre protection induced by the administration of a series of distinct antigens in experiments with these animals.
Campbell et al (1977) has focused the induction of protecting immunity by heterologous immunity. The study of this perspective showed that the infection of sheep by Cysticercus tenuicollis, the metacestode step of the measle (Taenia hydatigena) in dogs, produces partial protection against Fasciola hepatica. However, Hughes et al (1978) did not confirm this result. In other experiments, also was observed the incapacity of inducing protection with this cestode against Fasciola hepatica in experimental animals.
Adult and bisexual S. mansoni-infected mice developed a statistically significant resistance to Fasciola hepatica as well as against concomitant infections by both parasites resulting in a decreased amount of subsequent parasitic load and also a decreased number of eggs per adult worm (Christensen et al, 1978). The S. bovis-infected calves also showed some resistance to Fasciola hepatica and less marked liver damage (Sirag et al, 1981).
Pelley and Hillyer, 1978 & Hillyer and de Atica, 1980, have reported common antigens to Fasciola hepatica and Schistosoma mansoni found in Schistosoma eggs. The occurrence of false-positive reactions, in areas where both parasites are endemic, is another finding indicating cross reactivity and immunity. Hillyer, 1985 (Hillyer, G. V. 1985 “Induction of immunity in mice to Fasciola hepatica with Fasciola/Schistosoma cross-reactive defined immunity antigen”. Am. J. Trop. Med. Hyg. 34(6), pp. 1127-1131) and Hillyer et al, 1987 (Hillyer, G. V., Haroun, E. T. M., Hernandez, A. and Soler of Galanes, M. 1987. “Acquired resistance to F. hepatica in cattle using a purified adult worm antigen”. Am. J. Trop. med. Hyg. 37(2). pp. 363-369) have also demonstrated that an antigenic mixture derived from Fasciola hepatica can provide protection against subsequent infections caused by Fasciola hepatica and Schistosoma mansoni. 
Thus, it is believed, an effective vaccine will be the most powerful method, with a better cost/benefit relation, stopping the disease transmission and eradicating it from the human context regarding schistosomiasis; and from the veterinary context regarding fascioliasis.
A number of host species may develop partial resistance to Schistosoma mansoni starting from the initial infection or immunization with irradiated cercaria (Smithers, S. R. and Doenhoff, M. 1982. “Schistosomiasis” In: Immunology of Parasitic Infections. Blackwell Scientific Publications, 2nd Edition, Chapter 17, pp. 527-607). The state-of-the-art information, regarding the possibility of performing the immunization using raw extracts or material originating from Schistosoma mansoni parasitic forms (Clegg & Smith, 1978), has been making possible to produce a defined and effective vaccine against the parasite by using parasite antigens, i.e. non-living vaccines (Tendler, M. 1987. “S. mansoni: Protective antigens”. Mem. Inst. Oswaldo Cruz. Vol. 82. Suppl. IV. pp. 125-128). However, for most of the experiments using chemically defined and purified material, the greatest limitation was the incomplete degree of protection obtained in animals. As described by several authors and reviewed by Smithers, in 1982 (Smithers, S. R. 1982. “Fascioliasis and other Trematode Infections”. In: Immunology of Parasitic Infections. Blackwell Scientific Publications 2nd Edition, Chapter 17, pp. 608-621) there was already a consensus about the necessity for increasing the protection level induced in the experience-based immunoprophylaxis. However, it has been very difficult to settle a good animal model for the development of an effective schistosomiasis vaccine. The progress towards this target depends on the identification and refinement of highly effective antigenic molecules that could mediate the protecting immunity. (Tendler, M. “Schistosoma mansoni: Protective Antigens”, Mem. do Inst. Oswaldo Cruz. Rio de Janeiro, Vol. 82, Suppl. IV: 125-128, 1987).
In previous studies for finding antigens that mediate the schistosome-protecting immunity, the use of a complex mixture (called SE) of Schistosoma components early released by incubating adult living worms in buffered salt solution (Tendler, M. & Scarpin, M. 1979. “The presence of S. mansoni antigens in solutions used for storing adult worms”. Rev. Inst. Med. Trop. 21(6), pp. 293-296; Kohn et al, 1979). For the purpose of obtaining protection against cercaria infection through a vaccine, an experimental model was observed in two non-syngeneic animal hosts, with distinct susceptibilities to S. mansoni infection. One of them, the mouse, being susceptible and the other, the rabbit, partially resistant to infection.
It was possible to establish, in S. mansoni model of New Zealander rabbit, a reliable standard of percutaneous infection, with the recovery of homogeneous parasitic loads in number and size of parasites and the male/female ratio, during long term after the infection (Tendler, M., Lima, A., Pinto, R., Cruz, M., Brascher, H., Katz, N. 1982 “Immunogenic and protective activity of an extract of S. mansoni”. Mem. Inst. Oswaldo Cruz. Rio de Janeiro. Vol. 77(3), pp. 275-283; Tendler, M. 1985 and Tendler, M. 1986). Recent data suggest that the rabbit used as an experimental S. mansoni host may represent a new disease-immunity model.
Immunization experiments performed with rabbits using the mixture SE have resulted in very high protection levels after the challenge infection (Scarpin, M., Tendler, M. Messineo, L., Katz, N. 1980 “preliminary studies with a Schistosoma mansoni saline extract inducing protection in rabbits against the challenge infection”. Rev. Inst. Med. Trop. Sao Paulo. 22(4), pp. 164-172; Tendler, M. 1980; Tendler, M. 1982) (90% reduction in the average parasitic load of vaccinated animals compared to sex- and age-matched control animals infected with the same cercaria batch obtained from strain LE, a Brazilian S. mansoni strain). Besides presenting total protection against lethal infections, the SW (Swiss Webster) mice SE-immunized mice also showed significant protection when challenged with cercaria (Tendler, 1986). In order to assess the resistance, the vaccinated and challenged animals together with their respective controls, are subjected to venous perfusion of porta-hepatis and mesenteric systems for recovering and counting the adult parasitic load. The protection degree is calculated by the difference between the number of parasites recovered from controls compared to vaccinated animals (Tendler et al, 1982).
Based on “in vitro” evidence on the effectiveness of antibodies against distinct evolutionary parasite phases, in assays of eosinophilic or complement-dependent cytotoxicity (Grzych et al, 1982; Smith et al, 1982), the characterization of antigens recognized by the serum of knowingly resistant animals is used for identifying antigenic molecules potentially capable to mediate the protecting immunity (Bickle et al, 1986; Horowitz & Amon, 1985). Western blot experiments were performed to analyze the response from antibodies in SE-vaccinated rabbits. Testing the SE antigens against a panel of antisera of rabbits immunized with the same scheme (SE-CFA—Complex Mixture of the Components of the Complete Freund's Schistosoma-Adjuvant) the authors were capable to demonstrate, in immunoblots, the occurrence of two patterns of recognition of SE components.
Interestingly, the sera of rabbits that developed total protection only recognized some SE components. This result made possible to authors identify two SE-components groups: one group common to all individuals and other antigenic group only recognized by the serum of SE-vaccinated animals (rabbits) that were totally protected. These two recognition patterns were named pattern of “High” or “Low” protection and were used as “differential” antibodies. Based on both patterns of SE-component recognition by polyclonal sera of rabbits with different response to the same immunization scheme (probably due to the individual pattern of variation expected in non-syngeneic populations), a strategy for screening cDNA libraries in both sera was used. Taking into account the limitation of the incomplete knowledge about critical mechanisms of protecting response, both in experimental animals and in clinical schistosomiasis, the frequently adopted screening procedures comprise the use of either human sera from immune or susceptible subjects potentially infected (Carter & Colley, 1986) or monoclonal and polyclonal antibodies from immunized animals directed against several non-characterized antigens (Lanar et al, 1986; Balloul et al, 1987).
In initial attempts of molecular cloning of potential SE-protecting components, using differential screening, Drs. Klinkert, University of Heidelberg and Donnelson/Henkle, Iowa University, respectively, built two cDNA libraries of adult S. mansoni and S. japonicum worms. We can drawn a parallel from the immunoblot results, in which two distinct groups of clones were selected, corresponding potentially to the differential pattern of recognition of anti-SE rabbit sera with high and low protection. Parallel experiments, whose objective was to identify SE components, immunoblots of polyclonal rabbit anti-SE (high and low protection) sera were compared to rabbit sera, against purified Schistosoma paramiosine (provided by Dr. A. Sher, NIH). This protein had been defined as a molecule showing partial protection against Schistosoma mansoni infection of syngeneic mice (Lanar et al, 1986), its molecular weight is Mr(×10−3) 97, and it is sensitive to proteolytic degradation, resulting in two by-products of Mr(×10−3) 95 and 78 (Pearce et al, 1986).
The complex 97/95/78 was recognized by anti-SE sera with low and high protection and by monospecific serum against paramiosine. Besides the paramiosine, the high-protection, anti-SE sera have also recognized other peptides and proteins to be characterized and tested for their protection and immunological function.
The paramiosine detection, as a SE component, reinforces previous data from indirect immunofluorescence assays performed on section from adult Schistosoma worms, with rabbit's anti-SE serum, that reacted with the parasite surface and with the area among muscular layers (Mendonça et al, 1987), similarly to the demonstrated for the paramiosine (Pearce et al, 1986).
The result above was also concordant with the results from the screening of cDNA libraries performed as mentioned. Again the common paramiosine clones were isolated with anti-paramiosine and anti-SE sera. Clones recognized only by rabbit's anti-SE sera (high protection) were also obtained.
Among the other SE components having lower molecular weight, the 31/32-kDa component, described as potential candidate for schistosomiasis diagnosis, was also identified (Klinkert et al, 1987) and recently reported as a protease located in digestive tube, (Klinkert et al, 1988). These and other antigens identified in SE have shown to induce very low protection in vaccination testing.
For obtaining early released antigens from adult living worms (especially from secretion/excretion and tegument components), an incubation of adult living worms newly perfused in a chemically defined medium (PBS-Phosphate Buffered Saline), was used.
This strategy was adopted based on previous and frustrated attempts of other authors, aiming the induction of high resistance against Schistosoma infection from distinct raw S. mansoni extracts that could be theoretically depleted from relevant functional antigens. This premise was influenced mainly because the antigenic extraction procedures, commonly adopted by other authors, have used non-living parasites. Indeed, by using SE emulsified in CFA (Complete Freund's Adjuvant, as preferential adjuvant), administered by intradermal/subcutaneous route, high and long-term protection is reached in two experimental animal models against S. mansoni infection. The reason for using a rabbit model, that is uncommon in protection assays, was to reach an initial identification of potentially protecting antigens in partially resistant hosts (to be later tested in susceptible hosts) that, however, would be able to amplify the immune response and the effective mechanisms of parasitic death, once the rabbits are known as potent producer of antibodies.
Studies on immune response induced in vaccinated animals, aiming to identify functional, and relevant SE-protecting components, parasitic death site and mechanisms, as well as protection markers were the focus of our efforts in the last years. However, just recently, the information about SE composition, by using the identification and separation of their protecting components, became available.
The U.S. Pat. No. 4,396,000, published on Aug. 2, 1983, on behalf of Luigi Messineo & Mauro Scarpin (according to Reexamination Certificate 461 B1 U.S. Pat. No. 4,396,000 published on Feb. 11, 1986, the patent was revoked), describes an extract of adult Schistosoma mansoni worms, obtained by incubation in buffered sodium phosphate—sodium chloride—PBS 0.15 M (pH=5.8), comprising proteins, carbohydrates, and nucleic acids and/or by-products from these nucleic acids, separated in 4 main fractions by Sephadex G-100 and G-200 gel column chromatography. Immunodiffusion tests using rabbit whole anti-extract serum have shown 3 precipitation lines corresponding to fractions I and II and 1 to fractions III and IV. The rabbits immunized with the whole extract have developed total or partial resistance (at least 77%) against the subsequent challenge infection. The saline extract's antigenic material showed to be an effective vaccine in the treatment and immunization of schistosomiasis and other Schistosoma infections.
Illustratively, we emphasize that the U.S. Pat. No. 4,396,000 was revoked based on published articles. Among the data set corresponding to the antecedents of the present invention, we have the cloning and sequencing of a SE-derived component identified as being Sm14.
Moser et al (Moser, D., Tendler, M., Griffiths, G. and Klinkert, M. Q., have a published study “A 14 kDa Schistosoma mansoni Polypeptide is Homologous to a gene family of Fatty Acid Binding Proteins” Journal of Biological Chemistry vol. 266, No. 13, pp. 8447-8454, 1991). This study describes the gene sequencing and demonstrates the functional activity of the Sm14 as a fatty acid binding protein.
A complete nucleotide sequence that codifies the Schistosoma mansoni protein, Sm14, was obtained and determined from cDNA clones propagated in bacteriophage λgt 11 in Escherichia coli. The 14.8-kDa protein presents significant similarity, indicating homology, with a family of related polypeptide linked with hydrophobic ligands. Based on their affinity with long-chain fatty acids, members of this group of cytosolic proteins were originally identified. The purified recombinant protein showed affinity with fatty acids, in contrast with a mutant which lacks the first 16 N-terminal amino acids. The complete sequence of nucleotides can be described as a primer region beginning by ATG triplet at the height of nucleotides 123-125. The codifying region comprises 399 nucleotides, finishing in position 521. The protein of 133 residues from amino acids has molecular mass 14.847-kDa calculated based on its sequence.
Pérez, J. R. et al, (Perez, J>R., Medina, J. R. R., Blanco, M. A. G. and Hillyer. 1992. “Fasciola hepatica: Molecular Cloning, Nucleotide Sequence and Expression of a Gene Encoding a Polypeptide Homologous to a Schistosoma mansoni Fatty Acid-Binding Protein”. Journal of Experimental Parasitology, Vol. 74: No. 4, pp. 400-407) have proven that a polypeptide that presents cross reactivity with antiserum against immunoprophylactic Fh12 protein, shares significant homology with the Schistosoma mansoni 14.8-kDa protein, called Sm14, regarding amino acid sequence (Moser et al, 1991). In addition, it was proved that Fh12 is a potent immunogen and a molecule to be a candidate for immunoprophylaxis of both schistosomiasis and fascioliasis (Hillyer, 1985; Hillyer et al, 1987), as well as an important immunodiagnosis marker in the human fascioliasis (Hillyer et al, 1992). Moreover, the authors were trying to find a recombinant antigen containing Fh15 epitopes and such Fh15 portions could represent the same protein described as being Fh12.
Tendler et al have performed studies of protection against schistosomiasis in mice and rabbits with Sm14 recombinant protein. (1995, 1996). Therefore, the Sm14 protein cDNA was subcloned into pGEMEX-1 vector (Promega). The obtained construction, pGEMEX-Sm14, express Sm14 protein as a fusion with the product from gene 10 of bacteriophage T7 (major T7 capside protein), resulting in a chimeric protein with approximately 45 KDa. After the purification in SDS-PAGE preparatory gels, this fusion protein provided about 50% of protection against S. mansoni cercaria infection in experimentation animals, similar to the protection level reached by Saline Extract (SE) from these worms, used as positive control. On the other hand such recombinant protein provided 100% of protection against infection by Fasciola hepatica metacercaria (Tendler et al, 1995, 1996), showing that the Sm14 protein can be used as an anthelminthic vaccine. In addition, it is important to mention the U.S. Pat. No. 5,730,984, of the applicant's ownership.
However, during the storage of the Sm14 recombinant protein, the formation of a hardly-controlled precipitate was observed. Moreover, the obtaining of Sm14 recombinant proteins, according to the state-of-the-art, e.g. in pGEMEX, presents disadvantages because it is time-consuming and has low yield for large-scale production.
Thus, there is still a need of obtaining an antigenic material that can be obtained with high yield, in pilot scale, in compliance with GMP guidelines, preserving stability characteristics.